Peptides and peptide-containing molecules represent valuable tools for investigating biological systems, studying the binding and activity properties of biomolecules (e.g., enzymes, cell receptors, antibodies, kinases), exploring the etiopathological causes of diseases, and for validating pharmacological targets. Peptides and peptide-containing molecules are also attractive ligands for targeting protein-protein interactions and modulating the function of biological molecules such as enzymes and nucleic acids. The synthesis of combinatorial libraries of small peptides and peptide-containing molecules followed by screening of these chemical libraries in biological assays can enable the identification of compounds that exhibit a variety of biological and pharmacological properties. Bioactive peptides and peptide-containing molecules identified in this manner can constitute valuable lead compounds or facilitate the development of lead compounds towards the discovery of new drugs.
Both biosynthetic and synthetic methods are available in the art for preparing chemical libraries of peptides and peptide-containing molecules. Biological peptide libraries have been prepared, for example, by expressing a target polypeptide sequence fused to, or embedded within, a viral particle (e.g., phage display), a membrane receptor, or another protein scaffold in a host organism such as Escherichia coli or yeast, and randomizing the oligonucleotide sequence encoding for the target polypeptide sequence by random cassette mutagenesis or similar methods. These genetically-encoded peptide libraries can be used to identify peptide ligands with the desired property (e.g., high binding affinity toward a target protein) by displaying the peptide library on the surface of bacteriophage, bacteria or yeast and isolating the members of the library that bind to the target molecule by phage-panning, affinity separation, fluorescence-activated cell sorting or similar methods known in the art.
An advantage of these biosynthetic approaches is that very large collections of peptide ligands (108-1010 members or higher) can be generated within a short time and at low costs. After the screening step, the composition of the peptide ligands that exhibit the desired property can be rapidly determined by sequencing of the genetic elements that encodes for them. A potential limitation of these genetically encoded libraries, however, is that they rely on the combinatorial assembly of a relatively small pool of building blocks, i.e., naturally occurring amino acids, which limits the structural and functional diversity that these libraries can provide. Achieving a high degree of structural and functional diversity in a chemical library is crucial for increasing the likelihood of identifying a member within the library that exhibits the desired property or biological activity (e.g., high inhibitory activity toward a target enzyme or a protein-protein interaction).
Libraries of small peptides and peptide-containing molecules have been prepared synthetically by using solution- or solid-phase peptide synthesis in combination with combinatorial chemistry techniques. Split-and-mix methods and solid-phase peptide synthesis have been applied, for example, to prepare libraries of linear peptides where each members of the library is covalently bound to a resin bead (e.g., one-bead-one-compound library). Alternatively, arrays of peptides or peptide-containing molecules have been synthesized on glass slides, paper sheets, pins or other solid supports. These synthetic libraries can be screened for members displaying the desired activity by cleaving the peptide or peptide-containing molecule from the solid support and testing each member of the libraries in a biological assay. In some cases, the activity of the library members can be tested while they are still tethered to the solid support.
An advantage of these synthetic approaches is that a huge variety of diverse chemical structures can be used as building blocks in addition to the twenty natural amino acids. These alternative building blocks can include, but are not limited to, unnatural amino acids, peptoids, β- and γ-peptides, peptidomimetics, or amino acid-unrelated structures. These alternative scaffolds can be useful in conferring novel or improved conformational, binding or chemical/enzymatic stability properties to the peptide-based ligands not provided by naturally occurring amino acid structures. Compared to biological peptide libraries, a higher degree of structural and functional diversity is potentially accessible in libraries of synthetic peptide-based compounds and, in turn, this can facilitate the identification of compounds with the desired activity. However, sample handling and the need to spatially resolve each member of the library limits the number of molecules that can be prepared and screened in a productive and time-effective manner in the context of synthetic peptide libraries. In addition, unambiguous identification of the positive hits isolated during the screening step can be a laborious process faced with several pitfalls. This process has proven to be very challenging during the screening of large synthetic peptide libraries (>105).
While many peptides exhibit interesting biological activity, linear peptides do not generally represent suitable pharmacological agents as they are generally only poorly adsorbed, do not cross biological membranes readily, and are prone to proteolytic degradation. In addition, linear peptides fail to bind proteins that recognize discontinuous epitopes. The use of molecular constraints to restrict the conformational freedom of the molecule backbone can be used to overcome these limitations. In many cases, conformationally constrained peptides and peptide-containing molecules exhibit enhanced enzymatic stability, favorable membrane-crossing properties, and accessibility to structural analysis. Constraints that lock-in the active conformation of a peptide can also result in increased affinity due to the reduced conformational entropy loss upon binding to the receptor. Most therapeutically relevant peptides isolated from natural sources occur in cyclized form or contain intramolecular bridges that reduce the conformational flexibility of these molecules (e.g., immunosuppressant cyclosporin A, antitumor dolastatin 3 and diazonamide A, anti-HIV luzopeptin E2, and the antimicrobial vancomycin).
Various methods have been developed for preparing synthetic peptides and peptide-containing molecules in conformationally constrained configurations. Head-to-tail cyclic peptides can be prepared synthetically by cyclizing protected peptides in solution, by coupling the cyclization step with the removal of the cyclic chain from the solid support, or via ‘on-resin’ cyclization. Alternative cyclization strategies involve the use of native chemical ligation, photolabile auxiliary groups, or enzymes. Other strategies to restrict the conformational flexibility of linear peptides involve the formation of intramolecular bridges through the amino acid side chains, such as disulfide, lactam, oxime or alkenyl bridges.
While synthetic libraries of cyclic peptides can be prepared using these methods, the deconvolution of these libraries is faced with significant challenges. For example, Edman microsequencing of the active compounds isolated from the screening step cannot be carried out for cyclic peptides that lack free N-termini. Alternative methods (e.g., bead encoding/decoding with binary tags or MS/MS spectrometry) have been implemented to deconvolute libraries of cyclic peptides, but these procedures are complex, low-throughput, or they may not warrant unambiguous identification of the isolated compounds. Efficient and clean macrocyclization of peptide-based compounds can also be problematic. Altogether, these problems pose important constraints to the size of libraries of synthetic cyclic peptides that can be feasibly prepared and screened.
Methods for producing biological libraries of conformationally constrained peptides are also known in the art. For example, libraries of disulfide-constrained cyclic peptides have been prepared using phage display and fusing randomized polypeptide sequences flanked by two cysteines to a phage particle as described, e.g., in U.S. Pat. No. 7,235,626. Disulfide bridges are however potentially reactive and this chemical linkage is unstable under reducing conditions or in a reductive environment (e.g., inside a cell). Ribosomally produced peptides have also been constrained through the use of cysteine- or amine-reactive cross-linking agents. However, these methods rely on non-bioorthogonal reactions (e.g., cysteine-mediated alkylation or terminal/side-chain amine acylation) and thus bear the inherent risk of producing multiple undesired products via reaction of the cross-linking agents with multiple sites within the randomized peptide sequence or the carrier protein in a display system. Methods have also been described that allow for the preparation of head-to-tail cyclized peptides by using natural or engineered split inteins, as described in U.S. Pat. No. 7,354,756, U.S. Pat. No. 7,252,952, and U.S. Pat. No. 7,105,341. Similar to the biological libraries of linear peptides, however, the ribosomal nature of these compounds pose limitations to the chemical diversity of the ligand libraries generated through these methods. In addition, only head-to-tail cyclic ligand architectures can be obtained through these methods, which inherently limits the extent of structural diversity of the ligand libraries generated through these methods. Head-to-tail cyclic architectures also complicate the immobilization and isolation of these compounds for screening and identification purposes.
Citation or identification of any reference in Section 2, or in any other section of this application, shall not be considered an admission that such reference is available as prior art to the present invention.